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Doctoral Degrees (Botany)

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    Responses to chilling and cryopreservation of recalcitrant seeds of Ekebergia capensis from different provenances.
    (2020) Bharuth, Vishal Behary Lal.; Beckett, Richard Peter.; Pammenter, Norman William.
    Recalcitrant seeds are shed at relatively high water contents and are metabolically active. The effect of chilling the recalcitrant seeds of purportedly a single species, Ekebergia capensis, from Port Elizabeth (PE; Eastern Cape), St Lucia (KwaZulu-Natal [KZN]) and Tanzania (tropical southern Africa) was tested. Viability and axis ultrastructure, solute leakage and protein synthesis were investigated. Additionally, cryopreservation of embryonic axes (explants) was studied. In particular, the ability of cathodic water to improve explant survival was tested, and related to its effect on the production of potential harmful reactive oxygen species such as superoxide (·O2-) and hydrogen peroxide (H2O2), and its ability to maintain levels of total aqueous antioxidants (TAA). Shoot and root ultrastructure were examined after each cryopreparative step in explants treated with and without cathodic water, and ultrastructure correlated with survival. The seeds from PE retained 80% viability after 12 weeks storage at 1° and 3°C and 100% when stored at 6°C. Those from St Lucia were dead after 38 d storage at 3°C. All the seeds from Tanzania were dead after 9 d when stored at 3°C. The rate of protein synthesis decreased gradually over the storage period, irrespective of the provenance. Electrolyte leakage from axes showed that those from St Lucia and Tanzania ‘leaked’ solutes irrespective of the duration or temperature or storage, but those from PE showed an initial increase, which then decreased. A 30% mass loss was achieved after 48 d, 230 h and 21 h for seeds from PE, St Lucia and Tanzania, respectively. Nuclear ribosomal ITS1 sequences revealed the presence of three well-to-strongly-supported monophyletic clades corresponding to the geographical areas from which the seeds were sampled (PE, KZN and Tanzania). Axes from the seeds from St Lucia and Tanzania lost ultrastructural integrity during storage while those from PE did not. The levels of ·O2- and H2O2 increased gradually after each cryopreparative step. Using cathodic water allowed 30% of PE explants to survive cryopreservation, while none of the St Lucia explants did. Root ultrastructure was well preserved, however, gradual ultrastructural deterioration was observed in the shoot meristem.
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    The biology of pollination and seed dispersal in Clivia (Amaryllidaceae)
    (2019) Kiepiel, Ian.; Johnson, Steven Dean.
    Pollinator shifts have been implicated as drivers of angiosperm diversification. The hypothesis that a transition from bird to butterfly pollination took place in Clivia was tested, and floral traits investigated to determine which may have mediated the putative shift. Linking pollination systems with available phylogenies indicated a shift occurred from bird to butterfly pollination, accompanied by the evolution of upright trumpet-shaped flowers, scent emission and nectar volume reduction, whilst floral colouration and nectar chemistry remain unmodified. Results support the idea that pollinator shifts may explain major floral trait modifications during plant diversification. Breeding systems of Clivia were investigated, with the aims of demining the site and functional consequences of putative late-acting self-incompatibility (LSI). Results suggest that Clivia species are largely self-sterile as a result of LSI or severe inbreeding depression, but ovule discounting caused by self-pollination is not a major limitation on fecundity, and seed production appears to be mostly resource limited. Clivia miniata is pollinated virtually exclusively by butterflies. Functional significance of C. miniata floral traits were examined, with the aim of determining butterfly floral preferences and the functional basis of traits responsible for butterfly pollination. Colour is a key advertising signal, with orientation facilitating alighting, whilst size, scent, and shape also influence butterfly attraction. Dispersal mechanisms of numerous fleshy seeded Amaryllidaceae have been an enigma as seeds are unorthodox, toxic and unable to survive ingestion, yet packaged in brightly coloured fruits suggestive of animal dispersal. Dispersal and germination of Clivia miniata seeds was investigated. Results indicated consumption of fruit by primates which disperse seeds through non-ingestive spitting behaviour. The short distance of seed dispersal by primates is predicted to lead to restricted gene flow and genetic subdivision of populations. I conclude that shifts in pollination systems and the associated modification of suites of functional floral traits led to floral diversification in Clivia. Self-infertility in Clivia highlights pollinator dependence and pollination syndrome conformity reflects functional advertising signals. Gene flow appears to be governed by pollen flow and facilitated by pollinators rather than seed dispersal. Mating and breeding system evolution are likely a consequence of adaptation to isolated forest habitats.
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    The role of mutualisms in the evolution of flower and fruit traits in the Haemanthinae (Amaryllidaceae)
    (2022) Butler, Hannah Claire.; Johnson, Steven Dean.
    Diversification of flower and fruit traits are distinctive features of angiosperm radiation. Evolutionary shifts between different animal pollinator groups are usually accompanied by modifications in flower traits. Similarly, shifts between different animal seed dispersers are usually accompanied by modifications of fruit traits. The aim of this study was to assess the functional importance of flower and fruit traits in the African sub-tribe Haemanthinae (Amaryllidaceae) which consists of the closely related genera Scadoxus and Haemanthus. These genera occur in multiple habitats and exhibit a diversity of floral and fruit traits that are potentially related to their pollination and seed dispersal systems which has not been previously studied. The species of Scadoxus can be categorized according to two types of inflorescence architecture – ‘paintbrush’, where the reproductive parts of the flowers are tightly packed together, or ‘open brush’, where the reproductive parts are widely spaced. All Haemanthus species have ‘paintbrush’ inflorescences. I investigated the functional significance for pollination of these two inflorescence types. The genus Scadoxus appears to have undergone several shifts from butterfly to bird pollination. I found that both subspecies of S. multiflorus with open brush inflorescences are pollinated by butterflies and that S. puniceus and S. membranaceus with paintbrush inflorescences are pollinated by sunbirds. The system of butterfly pollination involves pollen being transferred from plant to plant via the surface of the butterfly’s wings. This system, previously thought to be unusual, is apparently common in the South African Amaryllidaceae and I speculated that nine species are pollinated this way. I found that S. multiflorus subspecies katherinae displays a system of late-acting self-incompatibility, whereby the tubes of self pollen are stopped at the ovary, as shown previously for other Amaryllidaceae. Self-incompatibility was also found for the sunbird pollinated S. puniceus. Intriguingly, S. membranaceus, which is very similar in appearance to S. puniceus, but rarely visited by sunbirds in its coastal forest habitat, was found to be self-compatible and capable of autonomous seed production. The genus Haemanthus, a sister clade to Scadoxus, occurs only in South Africa and Namibia, and consists entirely of species with ‘paintbrush’ style inflorescences. Haemanthus deformis is geoflorous with a very short peduncle and is pollinated by sunbirds that stand on the ground next to the inflorescence and bend over to feed on the nectar in the flowers. In the closely related H. albiflos, the inflorescence stem is longer and used as a perch. Both species have white flowers which is unusual for sunbird-pollinated plants. Haemanthus coccineus is found in the Cape Floral Region and has red flowers and bracts. This species has a much longer peduncle and is pollinated by sunbirds which grip onto the peduncle or bracts when feeding. H. humilis subsp. hirsutis is also visited by sunbirds which use the long peduncle as a perch when feeding on the pink flowers. Selective exclusion experiments indicated that H. humilis subsp. hirsutis is pollinated by both birds and insects, while H. coccineus and H. deformis are reliant on sunbirds. The tribe Haemantheae is defined by having fleshy, brightly coloured baccate fruits with large, recalcitrant seeds. No other species in the family have such a fruit type and the closest related tribe, Amaryllideae, have fruits characteristic of abiotic dispersal. S. multiflorus subsp. katherinae and S. puniceus occur in similar habitat of coastal to inland forested vegetation. I found that seeds of both taxa are dispersed by monkeys, which eat the fruits, depulping the seeds, and then spitting them out. In the genus Haemanthus, fruits are softer, and many species occurs in habitats without monkeys. I found that seeds of H. deformis are dispersed by birds and rodents which either depulp the seeds right next to the plant or disperse the seeds further away by carrying the fruits elsewhere. The seedlings require a shady microhabitat in bushclumps for survival and the dispersal system appears to favour either short distance dispersal into the immediate bushclump habitat or longer distance dispersal to different bushclumps. In conclusion, inflorescence and flower structure in the subtribe Haemanthinae play key roles in different pollination systems, with flowers in the paintbrush style inflorescences of Scadoxus puniceus and several Haemanthus species being pollinated by sunbirds, and flowers in open brush style inflorescence of S. multiflorus being pollinated by butterflies. Furthermore, the fruits of Haeminthinae are shown to be specialised for frugivory by various animals which discard the recalcitrant seeds. Mutualisms between various animals in Haemanthinae have therefore had an important impact on the evolution of flowers and fruit traits in this amaryllid subtribe.
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    The causes and consequences of Seriphium plumosum L. encroachment in semi-arid grassland communities of Gauteng province, South Africa.
    (2021) Pule, Hosia Turupa.; Tedder, Michelle Jennifer.; Tjelele, Julius Tlou.
    Abstract available in PDF.
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    Pollination and geographical divergence in flower colour of the drosera cistiflora species complex, or 'how the snotrosie got its colours'.
    (2019) Von Witt, Caitlin Greta.; Johnson, Steven Dene.
    The question of floral colour diversity among congeneric plants has intrigued evolutionary biologists since Lamarck—yet remains a hot topic amid the varied groundbreaking ecological insights that emerge to this day. I investigated potential causes of floral colour divergence in Drosera cistiflora sensu lato (Droseraceae), an insectivorous plant species complex which exhibits considerable variation over its geographical range in the Greater Cape Floristic Region of South Africa. Although several studies suggest that the foraging strategies of biotic pollination vectors can generate selective forces for floral trait diversification, no study has demonstrated unequivocally that pollinator-mediated selection is the core driver of shifts in flower colour. Indeed, selection by pollinating agents is not the sole possible explanation for floral colour disparity among populations, and other hypotheses, such as a role for edaphic factors, have been proposed as mechanisms modulating trends in flower colour. D. cistiflora s.l. is an exemplary study species complex for addressing these hypotheses as it displays remarkable heterogeneity in corolla colour—pink, purple, red, white and yellow—both between and within populations, which occur across a range of soil types. My primary aim was to establish whether pollinators can explain spatial patterns of flower colour in the complex. The thesis is divided into the following chapters: Chapter 1 is a general overview of the theory of pollinator-driven geographical divergence in floral traits, with a focus on flower colour, and includes a detailed account of the study species complex. Chapter 2 is an investigation into the breeding systems of D. cistiflora s.l., to assess whether floral attributes may reflect adaptations for allogamy. I discovered the complex to be highly pollinator-dependent for seed production with variable low autonomous selfing ability among floral colour forms and evidence for pollen limitation of fecundity. Chapter 3 examines associations between floral colour variation and the pollinating fauna and abiotic factors that may have played a role in the evolution of sympatric and allopatric floral colour forms. I show that respective forms are associated with geographically variable pollinator communities dominated by hopliine beetles (Coleoptera: Scarabaeidae: Hopliini), and that floral colour shifts appear to be largely independent of abiotic factors. These findings present a case for pollinator-mediated floral colour divergence in D. cistiflora s.l. In Chapter 4 I present the results of experiments using arrays (of models matched to D. cistiflora s.l. petal spectra and of reciprocally translocated flowering plants) that tested whether local pollinator discrimination among colours can explain geographical variation in flower colour. Analyses show that flower visitors discriminated significantly among models varying in colour and also among translocated flowers of D. cistiflora s.l. forms, and that the flower colour of the local D. cistiflora s.l. phenotype was generally favoured by insect visitors over introduced colours. Differential floral colour preferences and selection exhibited by polylectic pollinator communities across the range of study populations led me to conclude that the floral colour forms represent geographically divergent ecotypes adapted to broad pollinator assemblages. Chapter 5 specifically assesses the level of pollinator isolation between sympatric purple- and red-flowered D. cistiflora s.l. forms, to determine whether pollinators can maintain flower colour in the absence of macrogeographical barriers. Here, morphological observations and reproductive isolation indices demonstrate that strong pre-F1 barriers to gene flow between the two forms are governed largely by floral isolation, through differences in flower colour and beetle pollinator preferences. Evidence of early-acting postzygotic isolation is revealed by the significantly lower seed set from inter-colour hand cross-pollinations than from crosses within forms. Results support the taxonomic classification of endangered purple- and red-flowered D. cistflora s.l. populations above the rank of form and thereby signal their unprecedented conservation need. The thesis thus provides new evidence for adaptive floral divergence driven by a generalist pollinating fauna and demonstrates how the study of pollination ecotypes may benefit red-listing and conservation of threatened plant populations with poorly understood taxonomic limits. Lastly, in Chapter 6, I offer a summary of my main findings and their evolutionary, taxonomic and conservation significance, and outline key areas for further research.
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    Effects of antifungal treatments on some recalcitrant seeds.
    (2017) Makhathini, Aneliswa Phumzile.; Berjak, Patricia.; Pammenter, Norman William.
    Abstract available in PDF file.
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    Towards ameliorating some of the stresses associated with the procedural steps involved in the cryopreservation of recalcitrant-seeded germplasm.
    (2017) Naidoo, Cassandra Dasanah.; Berjak, Patricia.; Pammenter, Norman William.; Varghese, Boby.
    Abstract available in PDF file.
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    Automatic classification and ecological profiles of South-Western Transvaal Highveld grassland.
    (1973) Morris, Jeffrey William.; Villiers, T. A.
    Abstract available in PDF file.
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    Vitamin B-6 deficiency : biochemical and physiological changes with special emphasis on zinc status
    (1999) Pillay, Dhanabaikum.; Premjith, Gathiram.
    Abstract available on PDF file.
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    Biochemical, physiological and agronomic response of various sweet potato cultivars/varieties to drought stress in rainout shelters and field conditions.
    (2014) Laurie, Robert Naylor.; Van Staden, Johannes.; Finnie, Jeffrey Franklin.
    Drought is and will always be an issue in the cultivation of plants. Some plants have the ability to withstand a drought conditions to a certain degree while others, with other useful attributes, fail dismally. The value of testing genotypes for the ability to tolerate drought cannot be underestimated and will enhance the progress in the selection of drought tolerant genotypes. Thus, the objective of this study was to investigate the physiological, biochemical and agronomical reaction of sweet potato plants to drought and the procedures which could be used to test for sweet potato drought tolerance in the field. This was made possible through the creation of an environment at ARC-Roodeplaat in which sweet potato plants could be subjected to drought stress conditions. Thirty five sweet potato genotypes were planted in three trials in rainout shelters and open fields to analyze their physiological, biochemical and agronomical responses to drought stress. The majority of the genotypes were selected breeding lines with some cultivars from America, Peru and South Africa. These genotypes were chosen due to their range of traits for incorporation in crosses in the sweet potato breeding programme of the Agricultural Research Council (ARC). Drought stress conditions on the plants were induced through selective irrigation practices. In Trial 1 control plants were cultivated at field capacity while drought stressed plants received 60% and 30% of the amount of water of the control, respectively. In Trial 2, genotypes were planted in the field and under rainout shelters respectively. The field plantings acted as the control and received normal rain and irrigation while the rainout shelter planting received irrigation corresponding to 30% of field capacity. The plants in Trial 3 were subjected to control and drought conditions with the drought stressed plants receiving 30% of the water of the control. Leaf harvesting and phenotypical measurements were conducted twice during the trial period i.e. 60 and 120 days after planting. The drought stress impacted the growth of the sweet potato plants significantly. Canopy cover and stem length were severely influenced by the drought stress and resulted in huge declines of the respective values in all trails. Canopy cover values declined by more than twice compared to the control while stem length values were reduced by up to 10 times compared to the control. Antioxidant systems with particular reference to ascorbate peroxidase (AP), super oxide dismutase (SOD) and glutathione reductase (GR) reacted to the stress imposed and increased significantly. It was observed that values of the respective antioxidant enzyme systems increased sharply in the latter part of the trial and that the increase was also more intense at severe stress. The analysis of the antioxidant system made it possible to distinguish between the genotypes regarding their reaction to the stress. Results for carbon discrimination experiments in all the trials indicated that a significant decline in values took place as the drought stress increased. The decline appeared to be slightly more pronounced as the stress progressed. Also, as in the case of the antioxidant systems, it was possible to distinguish between genotypes even in the control treatments. The plants responded to the drought stress to the effect that a similar trend, (compared to the antioxidants), was observed with regards to stomatal conductance although genotypical differentiation was not possible in any of the stress conditions. It was demonstrated in the trials that the relative water content (RWC) values in the leaves of plants subjected to drought stress declined significantly between water treatments. Drought stress in the three trials had a severe impact on the nitrate reductase (NR) activity in the leaves of the plants. The decline in values were substantial but no significant differences could be detected between the genotypes except for the breeding line 2005-1-16 and cultivars Purple Sunset, Beauregard and Zapallo. Slight non-significant differences were observed between the genotypes at mild stress conditions but the severe stress conditions proved too harsh. Significant increases in the proline content of the sweet potato plants subjected to drought stress resulted in differentiation between the genotypes in Trial 1 and Trial 2, especially during the latter stages of the trials and at severe stress. Large reductions, up to 97%, of root yield were detected in the three trials. It appeared that the severe stress treatment proved too harsh to accomplish significant differences between the genotypes in all the trials. In Trial 1 the genotype Resisto differed significantly from the other genotypes and seemed to tolerate the drought the best in the mild stress conditions. Water use efficiency (WUE) values did allow for discrimination between the genotypes in Trial 1. A large decline in WUE values were observed in Trial 2 in general, although a few breeding lines 2005-7-4, 2006-4-4 and ix 2006-7-7 were prominent with high WUE values and could be recommended for use in a breeding programme. In Trial 3 the cultivar Bophelo and 199062.1 also exhibited higher WUE values which correlate well with yield data obtained from the same Trial. This could also prove valuable in the selection process. Due to the fact that multiple traits make a valuable contribution to the decision-making process in the selection for possible screening methods, statistical correlation was undertaken to establish possible relationships between traits. Good correlation was found between yield, stomatal conductance and WUE in Trial 1. This confirmed the assumption that a drop in stomatal conductance will result in lower root yield. Proline correlated also very well with the antioxidant enzyme levels of GR and AP which indicates that while the antioxidant enzymes play a role in combatting oxidants proline aid in possible prevention of moisture loss and stabilization of cell membrane structures. In Trial 2 good correlation was observed between yield, LAI, NR and CCI and to a lesser extent carbon-13 discrimination. This confirmed the belief that a decrease in LAI and CCI should have a negative effect on the yield due to less canopy cover and less chlorophyll for the capture of sunlight for photosynthesis. Results from Trial 3 also indicated good relationships between proline, GR and AP, as well as good relationships between yield, WUE, carbon discrimination and stomatal conductance (gs). It can hereby be concluded that the reaction of sweet potatoes to drought stress revealed results that can be of help for use in the future to successfully establish a protocol whereby successful selection of genotypes can be made in a biochemical, physiological and agronomical way. The study also provided proof that some of the approaches and procedures used in these trials can be successfully implemented in the drought screening of sweet potato.
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    Investigations on the biological effects of smoke-water and smoke-derived compounds in agriculture and horticulture.
    (2015) Papenfus, Heino Benoni.; Van Staden, Johannes.; Finnie, Jeffrey Franklin.
    Abstract available in PDF file.
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    The interaction between endophytic Fusarium species and Eldana saccharina (Lepidoptera) following in vitro mutagenesis for F. sacchari tolerance to control the borer in sugarcane.
    (2015) Mahlanza, Tendekai.; Rutherford, Richard Stuart.; Snyman, Sandra Jane.
    Eldana saccharina is a major pest in the South African sugar industry. Stalk damage by this borer and infection of bored tissue by opportunistic fungi result in loss of biomass and sucrose content, respectively. Amongst integrated management approaches, the best is employing E. saccharina-resistant genotypes. Resistance is attributed to physical stalk traits that impede boring and biochemical defences via nitrogen-based antiherbivory compounds. Further, in vitro assays have shown that Fusarium strains may be beneficial (e.g. F. pseudonygamai SC17) or antagonistic (e.g. F. sacchari PNG40) to the insect. The first objective of this study was, therefore, to establish the effect of sugarcane stalk traits and infection by Fusarium spp. on resistance to E. saccharina. In the first of two glasshouse trials, mature and immature stalk internodes of seven cultivars of known E. saccharina resistance ratings were inoculated with 2nd instar larvae via nodal wounds. Stalk rind hardness was greatest in both mature (42.2 units) and immature internodes (25 units) of the resistant cultivar N33. The softest of both mature and immature stalk regions were from the very susceptible N11 (32 units) and susceptible NCo376 (17.7 units), respectively. Percent fibre content in mature internodes was highest in the resistant N33 and N17 (12.8 - 14.2%) and lowest in the susceptible N11 and NC0376 (10.9 - 11.2%) cultivars. In all but one cultivar, % nitrogen content/dry mass was higher in immature internodes (0.65 - 1.2 %) than mature ones (0.36 - 0.91%) and lower in stalks of the resistant N41, N29 and N33 (0.36 - 0.75%) than in those of the susceptible NCo376 and N41 (0.48 - 1.27%) cultivars. Damage and mass gain by larvae retrieved from stalks were not entirely consistent with the cultivar resistance ratings, probably because the inoculation method by-passed the rind; N29 and N33 were unaffected by lack of rind protection. Hence, the tested stalk traits may contribute to E. saccharina resistance to varying extents in different sugarcane cultivars. In another trial, immature and mature stalks of NCo376 and N41 were inoculated with SC17 and PNG40 and then with E. saccharina larvae. The stalk area discoloured by Fusarium infection was smaller in the immature (6.1 - 7.1 cm²) than the mature (12.3 – 17.8 cm²) internodes. The smallest stalk length bored was in PNG40-infected NCo376 (3.3 cm) and N41 (1.7 cm) mature internodes, whilst NCo376 stalks colonised by SC17 (8.2 cm) were the most damaged. Hence, the proposal that Fusarium strains affect E. saccharina differently thereby impacting cultivar resistance/susceptibility to the borer, is supported. The in vivo activity of F. sacchari PNG40 against E. saccharina was also established, corroborating its potential as a biological control agent against the borer. As this application of PNG40 is impeded by the fungus being the causal agent of Fusarium stem rot in sugarcane, F. sacchari-tolerant plants were then produced via induced mutagenesis. Embryogenic calli of NCo376 and N41 were exposed to 32 mM ethyl methanesulphonate (EMS) for 4h. They were then placed on 100 ppm F. sacchari PNG40 culture filtrate (CF) at embryo maturation, germination or both stages, where 30.7 - 86.9% of the calli became necrotic and plantlet yield decreased by 59.2 - 99.2%. Roots of the regenerated plants were trimmed and placed on 1500 ppm CF. Plantlets with roots that regrew on CF medium beyond the 10 mm established threshold were deemed putatively tolerant (26.6 – 47.6% for EMS treatments, 5-24% for controls). These plants were acclimated and inoculated with PNG40 in the glasshouse. After 8 weeks, absence of symptoms, low lesion severity, re-isolation of PNG40 from the lesion and molecular identity of the isolates, confirmed some as PNG40 resistant. Re-isolation of PNG40 from undamaged tissue above the lesion, in plants with low lesion severity and no symptoms, confirmed endophytic colonisation and tolerance to the fungus in the mutants. Polymorphisms were detected in some mutants, using 24 RAPD primers. The use of the tolerant mutants in F. sacchari PNG40-mediated control of E. saccharina was then investigated. Stalks of five tolerant mutants and parents of each NCo376 and N41 cultivars were inoculated with PNG40 and with E. saccharina larvae, 3 weeks later. The length bored was less (1.0 - 4.7 cm) in stalks of PNG40 infected-mutants and parents than in the controls (3.9 - 9.0 cm). However, the % stalk discoloured area due to PNG40 infection was less in the mutants (10.6 - 22.0%) than in the parents (N41 - 28.9% and NCo376 - 30.2%). Re-isolation of PNG40 from undamaged tissue, within the inoculated internode and that above it, confirmed endophytic colonisation and fungal spread across internodes. Amongst stalks inoculated with PNG40, one mutant of NCo376 and two of N41 displayed limited boring (1 - 2 cm) and % discoloured area (10.6 - 15.1%), and the highest % of endophytically colonised stalk sections (50 - 75%) in the internodes immediately above those inoculated. There were no differences between the mutants and their respective parents in stalk rind harness, fibre and nitrogen contents. This work, therefore, resulted in the production of F. sacchari-tolerant mutants, demonstrated the toxicity of F. sacchari PNG40 against E. saccharina in vivo, and the ability of the PNG40-tolerant mutants to support endophytic colonisation by the fungus. Demonstration of these Fusarium - E. saccharina interactions in the mutants under field conditions will lead to the application of biological control of E. saccharina using PNG40, as part of integrated management approaches for the pest.
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    In vitro regeneration and secondary metabolites in cyrtanthus species.
    (2013) Ncube, Bhekumthetho.; Van Staden, Johannes.; Finnie, Jeffrey Franklin.
    Perennial geophytes form part of the diversified flora in southern Africa. The traditional medicinal system, integrates various plant components in the treatment of diverse ailments. In South Africa, bulbs are part of the extensively exploited floral resources for traditional medicine purposes. The reason for selective preference of certain groups of plant species or taxa, particularly geophytes, is rooted primarily on the beliefs that potent constituents are from the underground plant parts. The great surge of public interest in the use of plants as medicines however, assumes that plants will be available on a continuing basis. A vastly increasing human population coupled with the rapid degradation and fragmentation of natural habitats, exacerbate the threats posed by increasing demand on floral resources. The highly endemic members of the genus Cyrtanthus, most of which have limited geographic distribution ranges, are increasingly exploited for traditional medicines in South Africa. Bulbs of this species are the most preferred part for medicinal use, leading to the destructive harvesting of these plants. This form of plant harvesting poses threats to the long term sustainability of these plant resources in their natural habitats. Although sustainable harvesting of plant resources should be within the limits of their capacity for self-renewal, this seldom occurs owing to indiscriminate and destructive harvesting by commercial medicinal plant gatherers. As a consequence of this and other factors, intensive population decimation of a number of Cyrtanthus species is now evident and widespread, with some species threatened with extinction. The extinction of these species could lead to, in addition to the undesirable loss of genetic variability, loss of potential therapeutic agents. Conservation of these plant resources is therefore essential. The aim of this study was to establish efficient in vitro regeneration protocols for three threatened Cyrtanthus species (C. contractus, C. guthrieae and C. obliquus) endemic to southern Africa and explore the possible potential of improving the quality and quantity of bioactive secondary metabolites in culture. In vitro cultured twin-scale explants of the three selected Cyrtanthus species using different concentrations and combinations of 6-benzyladenine (BA) (0, 1.1, 4.4, 6.7, 8.9 μM) and naphthalene acetic acid (NAA) (0, 0.5, 1.1, 2.7 μM) in a 4 x 5 factorial treatment structure, established different optimal PGR combinations for shoot regeneration for each species. The highest shoot induction responses were obtained on MS medium with 4.4 μM BA/1.1 μM NAA for C. contractus and C. guthrieae and 6.7 μM BA/2.7 μM NAA for C. obliquus. The low concentration level of PGR requirements for shoot regeneration in C. contractus and C. guthrieae explants may suggest that the two species contain high enough endogenous hormones to induce shooting compared to those of C. obliquus. When the effect of different types and concentrations of cytokinins (CKs) [BA, kinetin (Kin), meta-topolins (mT), zeatin (ZT) and thidiazuron (TDZ)] on shoot multiplication were evaluated, 5 μM TDZ, 10 μM TDZ and 10 μM BA for C. guthrieae, C. contractus and C. obliquus respectively, were established as the optimum for shoot proliferation in each respective species. These results indicate, TDZ, a characteristically inexpensive CK, to be highly potent and effective in shoot proliferation of C. guthrieae and C. contractus. In terms of visual quality, shoots obtained from media supplemented with Kin and mT resulted in the best quality shoots in all three species at all concentrations tested. Furthermore, Kin also exhibited some auxin-like activitty by inducing rooting and callus on C. contractus and C. guthrieae cultures. The regenerated organogenic calli from C. guthrieae explants produced the optimum number of shoots through indirect organogenesis when transferred to MS medium supplemented with a combination of 0.1 μM picloram and 0.01 μM BA. An almost two-fold shoot proliferation frequency was obtained when the resulting callus-derived microshoots where subsequently transferred to the optimised shoot proliferation medium for the species. Regenerated shoots for all species were rooted successfully on half- and full-strength MS media without plant growth regulators, transferred to organic soil mix, and successfully acclimatised in greenhouse conditions. The developed micropropagation protocols provide a rapid and cost effective way of conservation, domestication and commercial cultivation of Cyrtanthus species. The levels of proline and polyphenolic compounds measured at intervals of three, four and five weeks from initial plantlet culture under different levels of salinity and osmotic regimes, increased in a stress-dependent pattern. The levels of these metabolites also showed a significant increase with an increase in the duration of plantlets under stress conditions. The highest proline concentration (9.98 μmol gˉ¹ FW) was recorded in C. contractus at 300 μM NaCl after five weeks. The high level of total polyphenolic compounds (147 mg GAE gˉ¹ DW) for the same species was however, recorded in the 150 μM NaCl stress treatment. The activity of proline dehydrogenase (PDH) (EC was shown to decrease with an increase in proline levels from week three to week five in almost all stress conditions evaluated. The high levels, particularly of phenolic compounds obtained under osmotic and salinity stress conditions in this study present a promising potential for manipulating culture and/or growing conditions for improved secondary compound production and hence medicinal benefits. In a study of the growth dynamics and patterns of assimilate partitioning to primary and secondary metabolites in response to varying levels and combinations of C (carbon) and N (nitrogen) in the culture media of Cyrtanthus guthrieae, relative growth rate (RGR) increased proportionally with an increase in C concentrations up to 88 mM sucrose (0.58 dˉ¹) beyond which it was hardly influenced by further increases in C. In C-limited media regimes with growth saturating N conditions, alkaloid accumulation became favoured while polyphenol content increased with an increase in C levels in the medium, a characteristic pattern that appeared to be less influenced by the amount of N. Of the primary metabolites, only proteins showed small significant variations across different media treatments, with starch and soluble sugars increasing proportionately with C levels. From a medicinal perspective, with regard to polyphenolic compounds in C. guthrieae, growth media conditions that allow for high levels of C pools in the tissue would thus be favourable for the enhanced synthesis of this group of compounds. The medium conditions with 175 mM sucrose and 10.3 mM NH₄NO₃ gave the highest total polyphenol, flavonoid and proanthocyanidin levels with a moderate growth rate. Pharmacological evaluation of the monthly collected C. contractus bulbs indicated some impressive bioactivities particularly the cytotoxicity effects against human cancer cell lines and enzyme inhibition (AChE and COX) by the extracts collected in certain months of the year. Of notable interest were the cytotoxicity effects, AChE and COX enzymes inhibitory activity of the extracts collected in May and September. Similarly, some extracts from in vitro precursor-fed plantlet and callus cultures demonstrated some excellent bioactivity, against COX and AChE enzymes. The results obtained from this study also reflect on the involvement of the environment in the quality of the extracts produced on a month to month basis and further suggest the importance of coinciding collection and use of plant extracts with the best time of the year or month. The good AChE and COX enzyme inhibitory activity by some of these extracts is of significant importance in the treatment of Alzheimer’s disease and neuroinflammation. The extracts represents an important component of traditional medicine.
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    Mosquitocidal activity against Anopheles arabiensis of plants used as mosquito repellents in South Africa.
    (2014) Mavundza, Edison Johannes.; Van Staden, Johannes.; Finnie, Jeffrey Franklin.; Maharaj, Rajendra.
    Ten plant species that are used as mosquito repellent in South Africa, Aloe ferox (leaves), Atalaya alata (leaves), Balanites maughamii (bark), Clausena anisata (leaves), Croton menyaarthii (leaves), Lippia javanica (leaves), Melia azedarach (leaves), Olax dissitiflora (bark), Sclerocarya birrea (seeds) and Trichilia emitica (seeds) were screened for adulticidal, larvicidal and repellent activities against Anopheles arabiensis, a potent malaria vector in South Africa. The plant extracts were screened following the WHO standard methods with slight modifications. The plant materials were extracted separately with ethanol (EtOH) and dichloromethane (DCM). All the extracts showed adulticidal activity. The highest activity was observed in the DCM extract of A. ferox leaves with an EC50 value of 4.92 mg/ml. With regards to larvicidal screening, all the DCM extracts showed larvicidal activity, while only five EtOH extracts showed activity. The highest larvicidal activity was found in the DCM extract of O. dissitiflora bark with an EC50 value of 25.24 μg/ml. All the investigated plants showed no repellent activity. Due to its good larvicidal activity, O. dissitiflora was evaluated for antibacterial, antifungal and antiplasmodial activities. The antibacterial activity was evaluated against two Gram-positive (Bacillus subtilis and Staphylococcus aureus) and two Gram-negative (Escherichia coli and Klebsiella pneumoniae) bacteria using the micro-dilution assay. The micro-dilution assay was also used to evaluate the antifungal activity of O. dissitiflora against Candida albicans. The antiplasmodial activity was evaluated against a chloroquine-sensitive strain of Plasmodium falciparum (D10) using the parasite lactate dehydrogenase assay. Both DCM and EtOH extracts showed good antibacterial activity against all four tested strains with MIC values less than 1 mg/ml. They also showed good antifungal activity with MIC values less than 1 mg/ml. Both DCM and EtOH extracts showed a moderate antiplasmodial activity, with IC50 values of 15.6 and 45 μg/ml, respectively. Good larvicidal activity observed in the DCM extract of O. dissitiflora bark prompted an attempt to isolate active compounds. Two active compounds were isolated from O. dissitiflora bark, ximeninic acid and a mixture of two closely related compounds (exocarpic acid and octadec-9,11-diynoic acid). The mixture of exocarpic acid and octadec-9,11-diynoic acid exhibited the highest larvicidal activity with an EC50 value of 17.31 μg/ml compared to ximeninic acid which had an EC50 value of 62.17 μg/ml. The results of the present study showed that the bark of O. dissitiflora and leaves of A. ferox may have potential to be used as larvicides and adulticides against An. arabiensis mosquitoes, respectively. This study also indicated that the bark of O. dissitiflora may have potential to be used as an antibacterial, antifungal and antimalarial agent.
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    Growth characteristics of three Eucalyptus clonal hybrids in response to drought stress : the underlying physiology.
    (2012) Eksteen, Alana.; Pammenter, Norman William.
    Previous research by Drew et al. (2009) on a SAPPI dendrometer trial, in northern KwaZulu-Natal, South Africa, yielded growth results for two Eucalyptus grandis clones (termed E. grandis x urophylla (GU) and E. grandis x camaldulensis (GC)). The GU clone was found to have a greater diameter than the GC clone, and further research has demonstrated that age, not environmental conditions, is the major determinant of tree growth for the GU and GC clone. The clones also showed different patterns of growth response following a rainfall event. In current study, young plants of three Eucalyptus grandis clones (two GU clones (GUA and GUW) and one GC clone) were grown in 80 L planting bags for 18 months at UKZN, Westville, South Africa. The experiments for this study entailed subjecting the three clones to three watering regimes. The trial was conducted using a randomised complete block (RCB) with a 33 factorial design (i.e. 9 treatments with 12 replicate plants in each treatment). The three watering regimes were monitored daily with a soil moisture probe and were a control (little or no water stress was applied), chronic water stress (mild, long-term, gradual water stress) and acute water stress (rapid, severe, cyclic water stress with periods of recovery from stress by re-watering). Physiological (photosynthesis, plant water relations and hydraulic conductance characteristics) and morphological (height, diameter and total biomass) measurements were performed. Two harvest periods determining Kh and total biomass at 9 and 18 months were undertaken, whereas morphological measurements were taken monthly throughout the trial. Considering that there were differing growth responses of clones in response to rainfall events (observed by Drew et al., 2009), the recovery of the plants from water stress was also studied (resistance to water flow in leaves, assimilation rates and stomatal conductance). Further investigation of leaf characteristics was performed to assess different aspects of the water transport system (stomatal density) and improvement of water use efficiency (WUE) in response to water stress by measurement of δ13C in leaf samples. The GC clone showed 30% greater height growth than the GU clones. Growth efficiency, root biomass and root:shoot were significantly greater in the GC clone. The GU clones showed significantly greater stem and leaf biomass, primarily due to the 25% greater total leaf area, after 18 months growth. Diameter of the plants subjected to the control, was 8% higher compared with water stress treatments (p = 0.036). Water stress significantly reduced tree volume by up to 10% and leaf area by 30%. Jmax and Vcmax were significantly lowered in plants subjected to acute stress at leaf wilting point (p < 0.001). After as little as 7 days re-watering however, Jmax and Vcmax were not different from the control. Plants subjected to chronic water stress showed moderately improved instantaneous WUE (8% increase compared with the control and acute stress). Long-term WUE (by measurement of δ13C in leaves, was significantly higher in leaves subjected to chronic water stress (p < 0.0001). Stomatal density was significantly different among clones, as the GUA clone showed complete stomatal absence on all upper leaf surfaces sampled (p < 0.001), although stomatal absence did not occur in leaves of the closely related GUW clone. Assimilation rate, stomatal conductance, Kh and total biomass were significantly positively correlated with one another. Recovery of plants subjected to acute stress differed among the GU and GC clones. An, gs and Rleaf (resistance to water flow in leaves) “recovered” (i.e. not significantly different from the control) by day 2 in the GC clone, but only by day 7 in the GU clones. There was hydraulic dysfunction in the GC clone which was suggested to be caused by collapse of the minor veins due to drought stress. The hydraulic dysfunction did not affect mesophyll tissue of the GC clone and thus hydraulic recovery was rapid. Although the GC clone was more drought tolerant (due to significantly greater root biomass), the selection of a GU clone would ensure improved wood productivity when planted commercially. The GUW clone showed enhanced traits of drought tolerance than the GUA clone including 20% less leaf dieback in response to water stress, as well as little to no variability of Kh in response to all watering regimes, and moderately improved WUE. Plants subjected to chronic stress showed long-term and instantaneous improvement in WUE, and greater diameters were maintained than plants subjected to acute stress. Perhaps the most important morphological and physiological parameter identified in the current study was that of leaf area. Leaf area differed significantly among eucalypt clones, in response to water stress and with tree age. Leaf area affected the expression of growth efficiency, hydraulic efficiency, total carbon assimilated and total biomass achieved. For the GU and GC Eucalyptus clones in the current study, the primary parameter driving physiological interactions and ultimately determining wood productivity could be considered to be leaf area.
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    Ultrastructure of the germinating Welwitschia mirabilis seed.
    (1975) Butler, Valerie.; Bornman, Chris H.
    The structure and chemical composition of quiescent Welwitschia mirabilis (Hooker fil.) seeds and resultant changes occurring during the first 7 days of germination were studied. Research was focussed on the megagametophyte and its interrelationship with the non-vascularised outgrowth produced by the embryo. This outgrowth was given the name "feeder" by Bower in 1881 who postulated that it functions as an absorptive organ. However the possibility existed that it merely fulfilled the mechanical role of anchorage. Following hydration activation of embryonic collar cells precedes that of gametophyte cells whose rate of activation is governed by relative distance from the embryo. This sequence of activation is suggestive of a stimulatory factor diffusing from the embryo into the gametophyte. Starch, protein and lipid reserves in the collar and developing feeder are consumed within 36 to 48 h. As a consequence the rapidly developing seedling is probably largely dependent on nutrient material in the gametophyte until the plumule emerges, after approximately 5 to 6 days germination. Ventral feeder cells in contact with gametophyte tissue apparently act as transfer cells, developing numerous small wall projections invested with plasmalemma which result in a much greater absorptive surface area. The large numbers of mitochondria occurring in these cells might suggest active uptake of nutrients. At the 3- to 4-day-stage the feeder and gametophyte adhere firmly. While this adherance probably facilitates translocation of nutrients it could also have the secondary function of anchoring the feeder in the gametophyte, thus providing the emerging plumule with a firm base. The apparent root cap origin of ventral feeder cells might explain the positive geotropism of the feeder, as recent work inter alia by Wilkins and Wain (1975) has shown that root cap cells may be geoperceptive. Cytochemical methods used at the light and electron microscope level suggest that reserve material within protein bodies of the embryo and gametophyte might exist as a proteincarbohydrate complex and that globoid origin might be cytoplasmic. The immediate digestion of protein body reserves in the embryo and gametophyte interface zone argues the presence of pre-existing hydrolytic enzymes laid down within the protein bodies prior to quiescence. However the enzymes responsible for reserve breakdown in deep gametophyte tissue seem to be synthesised de nova. Protein hydrolysis precedes lipid digestion which possibly indicates that some of the resulting free amino acids might be used in the de nova synthesis of lipases. Lipid bodies, microbodies, mitochondria and amyloplasts encircled with ER seem to form a complex. Fatty acids resulting from lipase action in the lipid bodies (Ching 1968) are probably converted by microbodies (glyoxysomes) to succunate (Breidenbach and Beevers 1967) which is converted to sucrose by the action of mitochondria (Cooper and Beevers 1969a, b). Excess sucrose is probably converted to starch and stored in the amyloplasts. In 5 days the mean dry mass of the gametophyte decreases by approximately 47% during which time the total amount of lipid decreases by 76.5% and protein by 14%. Although some of the hydrolysed fatty acids and amino acids are no doubt utilised in the gametophyte it is suggested that the majority of fatty acids are probably converted to sugars· which, together with free amino acids (and possibly simple peptides) are transported to and absorbed by the embryo via the feeder whence they are utilised for seedling growth.
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    'n Plantekologiese opname van Boschberg en sy omringende gebiede met spesiale verwysing na die weidingsfaktor.
    (1972) Van der Walt, Pieter Toxopeus.; Grunow, J. A.
    No abstract available.
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    Tissue culture studies on citrus and Welwitschia.
    (1972) Button, James.; Bornman, Chris H.
    Part I. IN VITRO CULTURE OF CITRUS EMBRYOS AND NUCELLAR ISOLATES Zygotic embryos of the Ellendale mandarin, a monoembryonic variety of citrus, were cultured on modified basal media of Murashige and Skoog (BM[1]), and White (BM[2]) , supplemented with various growth regulators and nutrient additives. The growth of immature embryos was greatly enhanced by the addition of 400 mg/l casein hydrolysate (CH) to the basal media. Coconut milk (CM) and malt extract (ME) enhanced growth to a lesser extent, while the addition of indoleacetic acid (IAA) and kinetin (KIN) at the concentrations used, was in no way beneficial. Nucellar isolates excised from abortive and normal Ellendale mandarin ovules eight to 20 weeks after anthesis, were cultured on BM[I] and BM[2] in the presence of various concentrations and combinations of IAA, indolebutyric acid, naph~haleneacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D), KIN, CM, benzyl adenine, 6-dimethylallylamino purine, yeast extract (YE), ME, CR, adenine (AD), adenine sulphate (AS), ascorbic acid (AA), and benzylthiazole-2-oxyacetic acid. Some of the isolates which remained alive for four months did develop callus but n~ differentiation of embryoids or other structures occurred. Unfertilized ovules from 8-12-week-old Washington Navel orange fruits provided nucellar isolates which were cultured on media similar to those upon which mandarin nucelli were unsuccessfully cultured. In the case of Navel orange nucelli however, BM[1] + 400 mg/l filter-sterilized ME, and BMl + 40 mg/l AD yielded numerous pseudobulbils which later developed embryoids. Adenine (10 mg/l) was more effective than 20 mg/l which in turn was more effective than 30 mg/l. Adenine was more effective than its equivalent amount supplemented in the sulphate form except at 10 mg/l where the two forms were equally effective. Zeatin (ZE) at 0,2 mg/l did induce some pseudobulbils and embryoids, but all these treatments were less effective than 400 mg/l ME. When transferred to BM[1] + GA[3] (1 mg/l) , embryoids developed roots and later, shoots. It was necessary to remove plantlets from the GA[3]-supplemented medium shortly after the first foliage leaves developed in order to prevent the development of weak, spindly plants . Plantlets were transferred from BM[1 ]+ GA[3] to BM[1] only, and then after careful conditioning they were planted out in soil. This appears to be the first successful attempt at inducing adventive embryogenesis 1n the nucellus of unpollinated, unfertilized citrus ovules in vitro. Part II. EMBRYO AND FREE-CELL CULTURE OF WELWITSCHIA MIRABILIS Welwitschia embryos, cultured on BMI supplemented with CR, and low levels of IAA and KIN, germinated and developed leaves but not roots. Embryos cultured on BM[I] with 5,0 and 10,0 mg/l NAA produced an abundance of friable callus from the hypocotyl root axis. This callus was used for starting suspension cultures aimed at inducing vegetative embryogenesis. A number of nutritional additives and hormones were used alone and in combination at various concentrations. Cells of numerous shapes and sizes were observed but no organogenesis was apparent in either suspension cultures or in cell colonies plated out on semi-solid agar media. A closer study of cell aggregates formed in suspensions supplemented with CM + 2,4-D revealed that internal division occurred in approximately 40 per cent of the larger cells. It is suggested that this internal division may constitute the first step in embryogenesis of Welwitsahia cells in suspension culture. It is also tempting to speculate that this process, which has been reported by other researchers, is the first step 1n embryogenesis of free cells in general. Although this attempt at inducing adventive embryogenesis in cell cultures of Welwitschia was unsuccessful, some encouraging results were obtained on potentially suitable media and possible initial stages in the organization of embryoids.
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    A plant ecological survey of the Umfolozi Game Reserve, Zululand.
    (1972) Downing, Brian Harvey.; Bayer, A. W.
    A landscape unit approach based on use of airphotos was used for investigation of Deciduous Acacia Woodland in the Reserve. The approach was useful for indicating that a quantity of as few as 531 samples could be suitable for sampling woody vegetation over the large, 493 km² area, and for showing where the samples might advantageously be distributed. Use of the approach minimised the extrapolation of community boundaries for mapping purposes. Normal association analysis of the samples revealed the nine woody consociations present and provided quantitative data on species constancy and fidelity. These data were used towards explaining low levels of homogeneity within consociations, as well as some close floristic similarities found between consociations. The successful emergence of a species to dominance in a consociation was ascribed to the presence of a particular, described soil series or rock substrate. The distribution pattern of the consociations resembled a soil-vegetation catena on the landscape. The consociations were grouped into defined physiognomic categories of Open, Closed and Riverine Woodland Associations that were distributed according to three soil associations. The effects of soil factors, fire and the biota on physiognomy, notably secondary thicket encroachment, were discussed; and the relationship between climate and phenology was mentioned. A quantitative description of the grass communities based on normal analysis of stratified sample data revealed a retrogression whereby climax grasses are being replaced by mid-seral and pioneer grasses. The retrogression was ascribed to selective grazing by an increasingly large biomass of enumerated, indigenous herbivores. Some of the management recommendations offered were based on empirically calculated estimates of the weights of dry grass required annually by the grazing animals. Copies of topography, place names, geology, land surface and vegetation maps are provided. The text is supplemented by check lists of plants and animals recorded, by eight figures, 33 tables and 54 photos.
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    Abscisic acid and other hormonal effects on growth in Spirodela.
    (1969) Van Staden, Johannes.; Bornman, Chris H.
    The effects of abscisic acid In particular, as well well as gibberellic acid and the cytoklnlns, 6-benzyladenine, kinetin, and 6-dlmethylal lalylamlnopurine, on the growth of Spirodela oligorrhiza were investigated. Abscisic acid effectively arrested growth permanently at concentrations down to 10¯¹ mg/I. Normal growth tended to be resumed at concentrations of 10¯² and 10¯³ mg/l between nine and twelve days after treatment. A concentration of 10[-8] mg/l, however, resulted in a significant increase in dry weight at both eight, nine and twelve days after introduction into the culture medium. It is suggested that the resumption of growth twelve days after treatment at those concentrations which inhibit growth up to nine days, was due to a possible progressive inactivation of abscisic acid resulting in a lowering of its concentration to a level that is promotive. It was furthermore found that the growth response of Spirodela in terms of dry weight production over a period of eight days is proportional to the log[10] concentration of abscisic acid. It is suggested that this curve can be used as a relatively reliable and easily performed bioassay to detect amounts of abscisic acid as low as 10[-5] μg. The assay is more reliable over the range 0.01 to 10,000 μg and appears not to be affected by gibberellin, benzyladenine and kinetin. The inhibitory effect of abscisic acid on growth in Spirodela was shown to be reversed by benzyladenine, kinetin and dimethylallalylaminopurine, although they were not equally effective in doing so. Benzyladenine at 1.0 mg/l was the most effective In overcoming growth inhibition by abscisic acid. Gibberellic acid, however, proved ineffective in reversing the inhibitory effect of abscisic acid on Spirodela oligorrhiza. The apparent Increases in growth obtained in some cases may have resulted more directly from gibberellic acid stimulation than from the Interaction of gibberel lie acid with abscisic acid.